RESUMEN
The let-7 family of miRNAs has been shown to be crucial in many aspects of biology, from the regulation of developmental timing to cancer. The available methods to regulate this family of miRNAs have so far been mostly genetic and therefore not easily performed experimentally. Here, we describe a small molecule screen designed to identify regulators of let-7 targets in human cells. In particular, we focused our efforts on the identification of small molecules that could suppress let-7 targets, as these could serve to potentially intercede in tumors driven by loss of let-7 activity. After screening through roughly 36,000 compounds, we identified a class of phosphodiesterase inhibitors that suppress let-7 targets. These compounds stimulate cAMP levels and raise mature let-7 levels to suppress let-7 target genes in multiple cancer cell lines such as HMGA2 and MYC. As a result, these compounds also show growth inhibitory activity on cancer cells.
Asunto(s)
MicroARNs/metabolismo , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genes Reporteros , Proteína HMGA2/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Inhibidores de Fosfodiesterasa/farmacologíaRESUMEN
Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results.
Asunto(s)
Algoritmos , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Expresión Génica , Redes Reguladoras de Genes/genética , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
It is clear that neural differentiation from human pluripotent stem cells generates cells that are developmentally immature. Here, we show that the let-7 plays a functional role in the developmental decision making of human neural progenitors, controlling whether these cells make neurons or glia. Through gain- and loss-of-function studies on both tissue and pluripotent derived cells, our data show that let-7 specifically regulates decision making in this context by regulation of a key chromatin-associated protein, HMGA2. Furthermore, we provide evidence that the let-7/HMGA2 circuit acts on HES5, a NOTCH effector and well-established node that regulates fate decisions in the nervous system. These data link the let-7 circuit to NOTCH signaling and suggest that this interaction serves to regulate human developmental progression.
Asunto(s)
MicroARNs/genética , Neuroglía/metabolismo , Células Madre Pluripotentes/metabolismo , Receptores Notch/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Inmunohistoquímica , MicroARNs/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuroglía/citología , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Células Madre Pluripotentes/citología , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Notch/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
Addition of a specific set of transcription factors reprograms somatic cell nuclei to a pluripotent state. Sequential addition of these factors, rather than the simultaneous exposure used in standard protocols, improves reprogramming efficiency. This sequential method favours a transition through a state with enhanced mesenchymal characteristics before driving an epithelial transformation on the way to the pluripotent state.